Yonsei Med J.  2013 Sep;54(5):1149-1157. 10.3349/ymj.2013.54.5.1149.

Sendai F/HN Viroplexes for Efficient Transfection of Leukemic T Cells

• Kim JS
• Lee YK
• Jeong HY
• Kang SJ
• Kim MW
• Ryu SH
• Kim HS
• Kim KS
• Kim DE
• Park YS

• KMID: 1793162
• DOI: http://doi.org/10.3349/ymj.2013.54.5.1149

Abstract

PURPOSE
Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells.
MATERIALS AND METHODS
The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a zeta-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR.
RESULTS
The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA.
CONCLUSION
This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines.

Keyword

Sendai F/HN protein; gene transfection; cationic Sendai viroplex; suspension cell

MeSH Terms

Cell Line, Tumor
HN Protein/genetics
Humans
Jurkat Cells
RNA, Small Interfering
Sendai virus/genetics
Transfection/*methods
Viral Fusion Proteins/genetics
Virosomes
HN Protein
RNA, Small Interfering
Viral Fusion Proteins
Virosomes

Figure

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  Fig. 1 Gel retardation analysis of cationic Sendai F/HN viroplexes encapsulating pDNA or siRNA. DMKE cationic liposomes and Sendai F/HN virosomes were complexed with pDNA (A) or siRNA (C) at various N/P ratios. CSVs and PCSVs were prepared at a 3 : 1 N/P ratio with pDNA (B) and a 6 : 1 N/P ratio with siRNA (D). All samples were analyzed on a 1% TAE agarose gel at 100 mV for 20 min. M; 1 kb DNA ladder. CSVs, cationic Sendai F/HN viroplexes; PCSVs, protamine sulfate-condensed cationic Sendai F/HN viroplexes.

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  Fig. 2 Viroplex-mediated luciferase transfection of Jurkat and CEM cells. Lipofectamine 2000 lipoplexes, DMKE lipoplexes, CSVs, or PCSVs containing pAAV-CMV-Luc or pEGFP (1 µg pDNA per well, 3 : 1 N/P ratio, 1 µg F/HN proteins) were added to Jurkat and CEM cells in a 24-well plate (each 1×106/well). After 4 h of transfection, cells were incubated for 24 h or 48 h, and luciferase expression in Jurkat (A) and CEM (B) cells was assayed. GFP expression in Jurkat (C) and CEM (D) cells was also assayed. Statistical analysis was done by Student's t test; *p<0.001 versus expression by DMKE lipoplexes, †p<0.001 versus expression by CSVs. RLU, relative light units; CSV, cationic Sendai F/HN viroplex; PCSV, protamine sulfate-condensed cationic Sendai F/HN viroplex; GFP, green fluorescence protein; pEGFP, plasmid encoding GFP; pAAV-CMV-Luc, plasmid encoding luciferase.

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  Fig. 3 Virosplex-mediated siRNA transfection of Jurkat and CEM cells. Jurkat (A) and CEM (B) cells were incubated with Lipofectamine 2000 lipoplexes, DMKE lipoplexes, or CSVs containing FITC-labeleled siRNA for 4 h or 24 h at 37℃. MFIs of transfected Jurkat (A) and CEM cells (B) were measured with flow cytometry. The same transfected Jurkat (C) and CEM (D) cells were stained with DAPI and then examined with a confocal laser microscope. Statistical analysis was done by Student's t test; *p<0.05 versus expression by DMKE lipoplexes. CSV, cationic Sendai F/HN viroplex; MFI, mean of fluorescence intensity.

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  Fig. 4 Viroplex-mediated vimentin siRNA transfection of Jurkat and CEM cells. Jurkat and CEM cells were transfected with Lipofectamine 2000 lipoplexes, DMKE lipoplexes or CSVs of vimentin siRNA (200 pmole). For comparison, cells were transfected by electroporation with 6 pulses of 10 msec duration at 320 V/cm. At 24 h after transfection, the levels of vimentin mRNA (A) in the transfected cells were quantified by RT-PCR analysis and compared with that of GAPDH mRNA, a positive control (B). Statistical analysis was done by Student's t test; *p<0.05 versus expression by DMKE lipoplexes. CSV, cationic Sendai F/HN viroplex.

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